PROJECT SUMMARY/ABSTRACT Myocardial infarction (MI) is one of the most common forms of cardiac injury. In a MI, coronary artery occlusion leads to a local restriction of blood and oxygen supply to the myocardium, causing many immediate and long-term changes throughout the myocardium that often manifest as arrhythmias or heart failure. Thus, a more detailed understanding of the remodeling processes that occur both local and distant to hypoxic injury are critically needed to develop new therapies for mitigating the progression to heart failure after MI. Recent studies have shown that hypoxia alters the cargo found in exosomes secreted by both cardiac fibroblasts and myocytes. This suggests that hypoxic and normoxic cardiac cell types could communicate via exosomes in post-MI myocardium. However, the effects of exosomes secreted by hypoxic cardiac cells on normoxic cardiac cells (and vice versa) is poorly characterized, in large part due to a lack of experimental tools. For example, the state-of-the-art for investigating hypoxia in vitro is to modulate oxygen globally with an incubator or hypoxia chamber. This approach does not mimic the oxygen gradients that are characteristic of post-MI myocardium and therefore precludes the investigation of ongoing cell-cell communication between normoxic and hypoxic cells, which could be a key mechanism of myocardial remodeling post-MI. We hypothesize that localized hypoxia affects the phenotypes of cardiac cells locally due to the direct effects of oxygen, and distally due to cell-cell communication between hypoxic and normoxic cells, mediated primarily by exosomes. To test this hypothesis, we will first fabricate a new microphysiological system that: (1) implements microfluidic gas supply channels to generate oxygen gradients; (2) has modular cell culture chambers to regulate cell-cell contact and paracrine signaling; and (3) integrates assays for quantifying cardiac fibroblast and myocyte structural and functional phenotypes, including existing ?Heart on a Chip? contractility assays previously advanced by the PI. We will then implement these devices for three Aims. In Aim 1, we will measure how oxygen gradients affect cardiac fibroblast phenotype, exosome RNA and protein cargo, and the activity of the oxygen-sensitive transcription factors, HIF-1 and HIF-2. In Aim 2, we will perform similar studies with cardiac myocytes and quantify functional phenotypes by integrating our ?Heart on a Chip? assays for measuring propagation velocity and contractile stress. In Aim 3, we will characterize cross-talk between hypoxic fibroblasts and normoxic cardiac myocytes, as well as hypoxic cardiac myocytes and normoxic fibroblasts. Together, our innovative microphysiological systems and rigorous experimental approaches will reveal significant new insights into the effects of localized hypoxic injury on the phenotypes of cardiac cell types, relevant to understanding myocardial remodeling post-MI. Our data will also establish new paradigms related to cell-cell communication pathways in hypoxia that are mediated by exosomes, which could be leveraged therapeutically. Additionally, our new devices could be used for medium-throughput screening of compounds for mitigating the effects of hypoxia.